An eco-friendly and cost-effective HPTLC method for quantification of COVID-19 antiviral drug and co-administered medications in spiked human plasma

The coronavirus-2 has led to a global pandemic of COVID-19 with an outbreak of severe acute respiratory syndrome leading to worldwide quarantine measures and a rise in death rates. The objective of this study is to propose a green, sensitive, and selective densitometric method to simultaneously quantify remdesivir (REM) in the presence of the co-administered drug linezolid (LNZ) and rivaroxaban (RIV) in spiked human plasma. TLC silica gel aluminum plates 60 F254 were used as the stationary phase, and the mobile phase was composed of dichloromethane (DCM): acetone (8.5:1.5, v/v) with densitometric detection at 254 nm. Well-resolved peaks have been observed with retardation factors (Rf) of 0.23, 0.53, and 0.72 for REM, LNZ, and RIV, respectively. A validation study was conducted according to ICH Q2 (R1) Guidelines. The method was rectilinear over the concentration ranges of 0.2–5.5 μg/band, 0.2–4.5 μg/band and 0.1–3.0 μg/band for REM, LNZ and RIV, respectively. The sensitivities of REM, LIN, and RIV were outstanding, with quantitation limits of 128.8, 50.5, and 55.8 ng/band, respectively. The approach has shown outstanding recoveries ranging from 98.3 to 101.2% when applied to pharmaceutical formulations and spiked human plasma. The method’s greenness was assessed using Analytical Eco-scale, GAPI, and AGREE metrics.


Materials and reagents
• Remdesivir reference substance (Purity 99.8% as certified) was generously gifted by EIPICo.10th of Ramadan City, Egypt.www.nature.com/scientificreports/ • Linezolid reference substance (Purity 99.8% as certified) was kindly provided by Averroes Pharma, 6th Industrial Zone Sadat City, Egypt.• Rivaroxaban reference substance (Purity 99.6% as certified) was kindly supplied as a gift sample from AChem- Block, Burlingame, USA.• HPLC grade methanol, ethanol and ethyl acetate were purchased from Fisher Scientifics, Belgium.
• Dichloromethane of analytical grade was obtained from Pioneers for chemicals (Piochem, 6th October City, Egypt).• Human plasma samples were supplied by Mansoura University Hospital, Mansoura, Egypt, and kept in the freezer at − 20 °C until usage.

Pharmaceutical dosage forms
Remdesivir-Rameda ® concentrate for solution for I.V infusion, batch number 214188 (Rameda Pharmaceuticals, 6th October City, Giza, Egypt) stated to contain 100.0 mg REM/vial, was obtained from the Egyptian market.Linezolid ® solution for IV infusion, batch number 21080023 (Global NAPI Pharmaceuticals, 6th October City, Giza, Egypt), was obtained from the Egyptian market and was noted to contain 200.0 mg/100 mL-vial.
Xarelto ® film coated tablets (Bayer Schering Pharma, batch no.04008500074763) were bought as well from the Egyptian market and stated to contain 10.0 mg/tablet of rivaroxaban.

Standard solutions
Accurately weighed amounts equivalent to 10.0 mg of REM, LNZ and RIV were put into three separate 10 mL volumetric flask then the volumes were brought up to the required volume by methanol for REM and LNZ and acetonitrile for RIV to produce 1.0 mg/mL standard solutions.The produced stock solutions remained stable for 14 days in the refrigerator without changing.

Chromatographic conditions
Following the saturation of the chromatographic jar for 30 min by the eluent system; dichloromethane-acetone (8.5:1.5, v/v).At 1.0 cm from the bottom edge, the sample bands were applied.Subsequently, chromatographic development and air drying was performed.This was followed by scanning the plates at 254 nm.

Calibration graphs construction
In methanol, REM, LNZ, and RIV concentration sets were prepared, with concentrations ranging from 20 to 550, 20 to 450, and 10 to 300 µg/mL, respectively.In triplicates, 10 µL aliquots of each flask were spotted on TLC plates to obtain concentration ranges of 0.2-5.5, 0.2-4.5, and 0.1-3.0µg/band of REM, LNZ, and RIV, respectively.By graphing concentrations (µg/band) versus the relevant areas under the peaks, calibration plots were constructed.

Pharmaceutical dosage forms analysis
An accurately measured volume of the Remdesivir-Rameda ® concentrate for solution for I.V infusion equivalent to 10.0 mg was transferred into a 10-mL volumetric flask, diluted with 5 mL methanol, shaken thoroughly, and then completed to the mark using the same solvent.
An accurately measured volume of Linezolid I.V solution equivalent to 10.0 mg was transferred into a 10 mL volumetric flask, 5 mL methanol was added, shaken well, and diluted to the required volume using the same solvent.
Accurately weighed ten Xarelto ® film-coated tablets were pulverized into fine powder then a weighed quantity equivalent to 10.0 mg of rivaroxaban was transferred into a 10 mL volumetric flask.5 mL of acetonitrile was added, and the solution was sonicated for 10 min.Using the same solvent, the volume was completed to 10 mL, then the solution was filtered using a 0.45 µm membrane filter.
To prepare working solutions, serial dilutions of each stock solution (1.0 mg/mL) were diluted with methanol.The corresponding regression equations for each drug were used to estimate the % recoveries of REM, LNZ, and RIV.

Spiked human plasma
Human plasma samples of 1.0 mL were transferred to 15 mL centrifuge falcon tubes, spiked with various concentrations of the studied drugs.After completing the volume to 5 mL with acetonitrile as a protein precipitating agent, the solutions were vortexed for 1 min before centrifuging at 3600 rpm for 30 min.Concentration ranges of 20-550, 20-450, and 10-300 µg/mL for REM, LNZ, and RIV, respectively, was obtained by transferring 1.0 mL of clear supernatants into 10 mL volumetric flasks and making up to the mark with methanol.REM, LNZ, and RIV concentration ranges of 0.20-5.50,0.20-4.50,and 0.1-3.00µg/band, respectively, were obtained by applying 10 µL aliquots of each concentration flask onto the TLC plates in triplicate.A blank experiment was run, and calibration graphs were generated concurrently.

Ethics approval
There are no human subjects in this research and informed consent is not applicable.The used plasma is pooled plasma from the blood bank of Mansoura university hospital.

Results and discussion
Rapid and economic TLC-densitometric technique was optimized to develop a validated, sensitive, and highly selective method for the estimation of REM, LNZ and RIV with minimal environmental damage.The proposed TLC approach has the plus of simultaneously determining multi analytes with a very straightforward sample preparation process and low solvent consumption.Notably the proposed method offers several advantages over previously reported traditional HPLC techniques 17,[29][30][31][32][33] , regarding its simplicity, cost-effectiveness, and rapid analysis time.Providing a promising alternative to conventional HPLC techniques for the estimation of REM in the presence of LNZ and RIV.Different chromatographic conditions were adjusted to achieve good separation with high resolution, sharp symmetric peaks and satisfactory R f values.

Method development and optimization
Different experimental parameters that affect the suggested TLC-densitometric technique such as mobile phase composition, saturation time, and scanning wavelengths, were adjusted.First, a mixture of petroleum ether and ethyl acetate in different ratios was examined.LNZ and RIV separated well, but RIV and REM separated poorly.
Testing the ethyl acetate-methanol combination with different ratios, only LNZ and RIV were well resolved but RIV and REM were slightly resolved.Upon replacing methanol with ethanol, the mixture afforded merely resolved peaks with poor resolution.
Upon testing dichloromethane-methanol only REM was eluted, and the others were retained.While dichloromethane-ethyl acetate resulted in poor separation between LNZ and RIV.On the flip side, dichloromethaneethanol has resulted in well resolved peaks however the REM peak has a tail.Thereby, acetic acid, formic acid, 25% ammonia solution, and triethylamine in different proportions were added to the tested mixture, however, poor resolution resulted.Eventually, a mixture of dichloromethane-acetone in different proportions was tested, yielding well-resolved sharp peaks with improved resolution.Therefore, mixture of dichloromethane-acetone in a ratio of 8.5:1.5 v/v was used throughout this approach.
Saturation times ranging from 15 to 45 min, were investigated since they had a considerable impact on chromatographic separation.With a saturation time of 30 min, satisfactory results were obtained.Different scanning wavelengths (235, 254, 280 nm) were tested, and 254 nm demonstrated the best sensitivity, yielding sharp, symmetrical peaks with minimal noise.

Method validation
ICH Q2(R1) recommendations have been implemented to evaluate the method's validation criteria 34 , including:

Linearity and range
Under the above mentioned densitometric procedures, the regression plots were generated over the concentration range of 0.20-5.50,0.20-4.50and 0.10-3.00μg/band for REM, LNZ and RIV, respectively.Good correlation coefficients were obtained, and linear regression equations were as follow: where, y is the peak area at 254 nm, x is the concentration in µg/band, and R 2 is the correlation coefficient.As illustrated by Table 1, the suggested technique has good linearity.

Accuracy
To evaluate the accuracy of the suggested method, the percentage recoveries of six concentrations of analytes within the linearity range were analyzed in triplicate.The obtained data has shown that the proposed method has a high degree of reliability and accuracy with small values of standard deviation (Table 2).

Intra-and inter-day precision
The proposed method's intra-and inter-day precision was investigated.For intra-day precision, three different concentrations of each drug were measured in triplicate on the same day, and three successive days for interday precision.This was supported by the relative standard deviation (%RSD) values which were less than 2%, as shown in Table 3.

Detection and quantitation limits
The method's sensitivity was evaluated by calculating detection and quantitation limits using the standard deviation of the intercept (σ) and the average slope (S); where LOD; (3.3*σ)/S and LOQ; (10*σ)/S, respectively.The resulting limits for REM, LNZ, and RIV are shown in Table 1 confirming that the proposed method has good sensitivity.

Robustness
Minor but intentional changes were made to the chromatographic technique parameters, and the percentage relative standard deviation (% RSD) was used to assess the robustness.Slight alteration was done in the proportions of the mobile phase system; dichloromethane, acetone (8.5:1.5 ± 0.10 mL), and saturation time (30 ± 5 min).The findings demonstrated the approaches' reliability and robustness, demonstrating that the tested factors had no apparent impact (Table 4).

System suitability testing
System suitability tests showed good results compared to reference values for parameters like capacity factor, tailing factor, and resolution 35,36 .(Table 5).www.nature.com/scientificreports/

Application to pharmaceutical formulations
The pharmaceutical formulations of REM, LNZ, and RIV were successfully estimated employing the suggested technique.The obtained percentage recoveries were satisfactory.Using the variance ratio F-test and the student's t-test 37 , the results were compared to previously reported methods 17,38,39 .No significant differences were found, demonstrating the absence of excipient interference.The findings were acceptable, proving the proposed method's high accuracy, Table 6.

Application to spiked human plasma
In human plasma, the new technique showed great sensitivity.The outcomes indicate that the method can accurately detect the investigated drug concentrations in human plasma without any interference, as shown in Fig. 3 and Table 7.

Green assessment of the proposed method
Green analysis relies on the use of green chemicals, limited usage of hazardous ones with no waste production and reduced energy consumption.Eco-Scale technique, the Green Analytical Procedure Index (GAPI) metric, and the Analytical GREEnness metric approach and Software were investigated to evaluate the suggested TLCdensitometric method's greenness [40][41][42] .The green profiles for the proposed method using the mentioned metrics are shown in Table 8.

Analytical eco-scale assessment of the proposed method
Analytical Eco-scale is an easy-to-implement technique for quality control laboratory work.The analytical Eco Scale score is computed using the following formula: analytical Eco Scale score = 100-∑ penalty points.It is determined by allocating penalty points to each of the parameters of the outlined technique, including the amount of reagents, occupational hazards waste, and energy 40 .The analytical approach is recognized as an outstanding

GAPI assessment of the proposed method
A relatively recent method for gauging greenness is the green analytical process index (GAPI), created by Plotka-Wasylka 41 .It follows the entire process, from sample collection through waste processing.It presents 15 items to be examined using three degrees of color: green, yellow, or red, where red represents bad impact, yellow for intermediate, and green for safe and low environmental dangers.This allows for a thorough examination of each stage in the analytical procedure.The proposed technique fulfilled the majority of GAPI criteria with only 3 red regions.The first was caused by offline sample collection, the second by using a non-green solvent to precipitate plasma proteins, and the third by the absence of waste treatment possibilities.There are four yellow-colored regions corresponding to region 5, 10, 11 and 14.Region 5 is yellow as the method requires simple preparation such as filtration, while regions 10,11 are yellow because the solvents used have moderate health and environmental effects, according to the National Fire Protection Association (NFPA), and region 14 is yellow as the waste  www.nature.com/scientificreports/produced is less than 10 ml per sample.Overall, the results show that the suggested approach is environmentally friendly, as shown in Table 8b.

AGREE assessment of the proposed method
Recently, the AGREE assessment tool was reported.The assessment is carried out with ease, implementing user-friendly software with an automatically created graph, and an evaluation report 42 .AGREE provides a clockshaped graph with a perimeter divided into 12 sections based on the 12 tenets of green analytical chemistry.To assess whether the analytical technique complies with the green analytical chemistry (GAC) concept, each division is assigned to a different color on a red-yellow-green scale.A color and a number representing the overall evaluation on a scale of 0 to 1 are located in the center of the AGREE graph.The proposed method scored 0.81 as demonstrated in Table 8c, which indicates the greenness characteristics of the developed method.

Conclusion
A simple, selective, and accurate chromatographic method has been developed for simultaneous determination of REM, LNZ and RIV in spiked human plasma.As multiple samples can be run simultaneously using minimal volumes of solvents, the TLC-densitometric approach saves time and lowers the cost of analysis.Moreover, the proposed method is eco-friendly with low impact on the environment.It has the benefits of being very selective, sensitive, and reproducible.It provides a fast and validated TLC analytical method for measuring remdesivir in the presence of linezolid and rivaroxaban for simple therapeutic medication monitoring and possible pharmacokinetic investigations.
Table 8. Green Assessment of the proposed TLC method by Analytical Eco-scale, GAPI and AGREE.

Table 1 .
Analytical performance data for the determination of the studied drugs by the proposed method.

Table 2 .
37curacy evaluation for the estimation of REM, LNZ, RIV by the proposed method.Each result is an average of three separate determinations.The tabulated t and F values are 2.36 and 5.78, respectively, at p = 0.05, Ref.37.

Table 3 .
Precision results for the determination of the studied drugs by the proposed TLC method.

Table 4 .
Robustness testing of the proposed method.

Table 5 .
System suitability parameters of the proposed method.greenanalysisif the score is higher than 75.The designed TLC-densitometric technique has an Eco-Scale score of 91, as shown in Table8a, indicating that the outlined analytical procedure is considered green.

Table 6 .
37timation of the investigated drugs in their pharmaceutical dosage form with statistical comparison to previously reported methods.Each result is an average of three separate determinations.The tabulated t and F values are 2.77 and 19, respectively, at p = 0.05, Ref.37.

Table 7 .
Application of the proposed method to the determination of REM concurrently with LNZ and RIV in spiked human plasma.